Hexyl butyrate

Endpoint summary

Description of key information

Studies performed to recognised testing guidelines with GLP.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 April 2018 to 03 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

04 Feb 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

This is a Non in vivo test and the test material is used in cosmetic ingredients. Regulation 1223/2009 Article 18 restricts the use of in vivo studies on these types of raw materials.

Specific details on test material used for the study:

Appearance: Clear colourless liquid
Purity/Composition: See Certificate of Analysis
Test item storage: At room temperature protected from light

Details on the study design:

MATERIALS AND METHODS
Test Item and Reference Item
Test Item (Hexyl Butyrate)
Identification: Hexyl Butyrate
Appearance: Clear colourless liquid
Batch: 202509
Purity/Composition: See Certificate of Analysis
Test item storage: At room temperature protected from light
Stable under storage conditions until: 16 September 2018 (expiry date)
Additional information
Test Facility Test Item Number: 209412/A
Purity/Composition correction factor: No correction factor required
Chemical name (IUPAC, synonym or trade name: Hexyl butanoate
CAS number: 2639-63-6
EC number: 220-136-6
Molecular structure:
Molecular formula: C10H20O2
Molecular weight: 172.26 g/mol
For Certificate of Analysis see Appendix 6.

Reference Item (Positive Control Cinnamic Aldehyde)
Identification: Cinnamic aldehyde
Test Facility Test Item Number: RS473/A
Appearance: Yellow liquid
CAS Number: 104-55-2
Molecular Structure:
Molecular Formula: C9H8O
Molecular Weight: 132.16 g/mol
Batch: MKBP1014V
Purity: 98.4%
Test item storage: In the refrigerator (2-8°C)
Stable under storage conditions until: 31 May 2018
Supplier: Sigma-Aldrich Chemie GmbH, Steinheim, Germany
Purity/composition correction factor: Yes
For Certificate of Analysis see Appendix 6.

Test Item Characterization
The Sponsor provided to the Test Facility documentation of the identity, purity, composition, and stability for the test item. A Certificate of Analysis was provided to the Test Facility and is presented in Appendix 6.

Reserve Samples
For each batch (lot) of test item, a reserve sample (about 0.5 gram) was collected and maintained under the appropriate storage conditions by the Test Facility and destroyed after the expiration date.

Test Item Inventory and Disposition
Records of the receipt, distribution, and storage of test item were maintained. With the exception of reserve samples, all unused Sponsor-supplied test item will be discarded or returned to the Sponsor after completion of the scheduled program of work. Records of the decisions made will be kept at the Test Facility.

Dose Formulation and Analysis
Preparation of Test Item
No correction for the purity/composition of the test item was performed.
Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ), ACN:MQ (1:1, v/v), isopropanol, acetone:ACN (1:1, v/v) and dimethylsulfoxide (DMSO):ACN (1:9, v/v).
Test item stock solutions were prepared freshly for each reactivity assay.
For both the cysteine and lysine reactivity assay 32.17 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1868 µL ACN after vortex mixing to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.
Any residual volumes were discarded.

Test System
Test system Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH).
The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL.
Rationale Recommended test system in the international OECD guideline for DPRA studies.
Source JPT Peptide Technologies GmbH, Berlin, Germany.
Batch See Appendix 7 for detailed information.
Storage The peptides were stored in the freezer (≤-15°C) for a maximum of 6 months.

Reagents
Acetone HiPersolv, VWR international, Amsterdam, The Netherlands
Acetonitrile (ACN) HPLC grade, Fisher Chemicals, Loughborough, England
Ammonium acetate Fractopur, Merck, Darmstadt, Germany
Ammonium hydroxide 25%, Merck
Dimethylsulfoxide (DMSO) Seccosolv, Merck
Disodium hydrogen phosphate
(Na2HPO4·12H2O)

Emsure, Merck
Isopropanol LiChrosolv, Merck
MilliQ-water (MQ) Tap water purified by reversed osmosis and subsequently passed over activated carbon and ion exchange cartridges; Millipore, Bedford, MA, USA
Sodium dihydrogenphosphate dehydrate
(NaH2PO4·H2O)

Emsure, Merck
Trifluoroacetic acid (TFA) >99%, Sigma Aldrich, Zwijndrecht, The Netherlands

Experimental Design
Preparation of Solutions for Cysteine Reactivity Assay
Synthetic Peptide Containing Cysteine (SPCC) Stock Solution
A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes
followed by 5 minutes sonication.

SPCC Reference Control Solutions
Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCC stock solution with 250 µL
ACN.

SPCC Calibration Curve
A SPCC calibration curve was prepared as described in the table below.

Preparation of SPCC Calibration Curve
SPCC calibration solutions SPCC concentration (mM) Preparation
STDcys1 0.534 1600 µL stock solution of 0.667 mM SPCC + 400 µL ACN
STDcys2 0.267 1 mL STDcys1 + 1 mL STDcys7
STDcys3 0.133 1 mL STDcys2 + 1 mL STDcys7
STDcys4 0.067 1 mL STDcys3 + 1 mL STDcys7
STDcys5 0.033 1 mL STDcys4 + 1 mL STDcys7
STDcys6 0.017 1 mL STDcys5 + 1 mL STDcys7
STDcys7 0 8 mL phosphate buffer (pH 7.5) + 2 mL ACN

Co-elution Control, Test Item and Positive Control Samples
The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described in the table below.
Preparation of Co-elution Control, Test Item and Positive Control Samples
Sample Replicates Sample code Preparation
Co-elution control (CC) 1 CCcys-209412/A 750 µL Phosphate buffer pH 7.5
200 µL ACN
50 µL 209412/A test solution (100 mM)
Cinnamic aldehyde (PC) 3 PCcys-1 to PCcys-3 750 µL Stock solution of 0.667 mM SPCC
200 µL ACN
50 µL Cinnamic aldehyde solution
(100 mM in ACN)
Test item 3 209412/A-cys-1 to 750 µL Stock solution of 0.667 mM SPCC
209412/A 209412/A-cys-3 200 µL ACN
50 µL 209412/A test solution (100 mM)

Preparation of Solutions for Lysine Reactivity Assay
Synthetic Peptide Containing Lysine (SPCL) Stock Solution
A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10 mg of SPCL in 19.31 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.

SPCL Reference Control Solutions
Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL ACN.

SPCL Calibration Curve
A SPCL peptide calibration curve was prepared as described in the table below.

Preparation of SPCL Calibration Curve
SPCL calibration solutions SPCL concentration
(mM) Preparation
STDlys1 0.534 1600 µL stock solution of 0.667 mM SPCL + 400 µL ACN
STDlys2 0.267 1 mL STDlys1 + 1 mL STDlys7
STDlys3 0.133 1 mL STDlys2 + 1 mL STDlys7
STDlys4 0.067 1 mL STDlys3 + 1 mL STDlys7
STDlys5 0.033 1 mL STDlys4 + 1 mL STDlys7
STDlys6 0.017 1 mL STDlys5 + 1 mL STDlys7
STDlys7 0 8 mL ammonium acetate buffer (pH 10.2) + 2 mL ACN

Co-elution Control, Test Item and Positive Control Samples
The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described in the table below.
Preparation of Co-elution Control, Test Item and Positive Control Samples
Sample Replicates Sample code Preparation
Co-elution control (CC) 1 CClys-209412/A 750 µL Ammonium acetate buffer pH 10.2
250 µL 209412/A test solution (100 mM)
Cinnamic aldehyde (PC) 3 PClys-1 to PClys-3 750 µL Stock solution of 0.667 mM SPCL
250 µL Cinnamic aldehyde solution
(100 mM in ACN)
Test item 3 209412/A-lys-1 to 750 µL Stock solution of 0.667 mM SPCL
209412/A 209412/A-lys-3 250 µL 209412/A test solution (100 mM)


Sample Incubations
After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 24.5 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
Prior to HPLC PDA analysis the samples were visually inspected for precipitation. The samples that showed a phase separation were centrifuged (at 400 g) for 5 minutes at room temperature.

HPLC-PDA Analysis
SPCC and SPCL peak areas in the samples were measured by HPLC PDA. Sample analysis was performed using the following systems:

System 1 (used for Cysteine Reactivity Assay):
• Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
• MPS 3C autosampler (DaVinci, Rotterdam, The Netherlands)
• LC Column oven 300 (Thermo Scientific)
• Surveyor PDA detector (Thermo Scientific)

System 2 (used for Lysine Reactivity Assay):
• Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
• HTC PAL autosampler (DaVinci, Rotterdam, The Netherlands)
• Column Oven #151006 (Grace, Worms, Germany)
• Surveyor PDA detector (Thermo Scientific)
All samples were analyzed according to the HPLC-PDA method presented in Table 1 (Appendix 1). The HPLC sequences of the cysteine and lysine reactivity assay for the test item are presented in Table 2 (Appendix 1).

ACCEPTABILITY CRITERIA
The following criteria had to be met for a run to be considered valid:
a) The standard calibration curve had to have an r2>0.99.
b) The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL.
c) The maximum standard deviation (SD) for the positive control replicates had to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion.
d) The mean peptide concentration of Reference Controls A had to be 0.50 ± 0.05 mM.
e) The Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN had to be <15.0%.

The following criteria had to be met for a test item’s results to be considered valid:
a) The maximum SD for the test item replicates had to be <14.9% for the Percent Cysteine Depletion and <11.6% for the Percent Lysine Depletion.
b) The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50±0.05 mM.

Positive control results:

The results of the positive control cinnamic aldehyde are presented in Table 7 (Appendix 3). The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 74.5% ± 0.9%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

Key result

Parameter:

other: SPCC depletion

Value:

0.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Parameter:

other: SPCL depletion

Value:

0.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

RESULTS

Solubility Assessment ofthe Test Item

At a concentration of 100 mM, Hexyl Butyrate was not soluble in MQ and ACN:MQ (1:1, v/v), but was soluble in ACN, acetone:ACN (1:1, v/v), DMSO:ACN (1:9, v/v) and isopropanol.

Solubility of the 100 mM test item solution prepared in ACN, acetone:ACN (1:1, v/v), DMSO:ACN (1:9, v/v) or isopropanol was investigated in the SPCC assay buffer by mixing 50 µL of the 100 mM test item solution with 750 µL phosphate buffer pH 7.5 and 200 µL ACN followed by vortex mixing. For all four solvents, formation ofa phase separation in the form of an oily top layer was observed.

Solubility of the 100 mM test item solution prepared in ACN, acetone:ACN (1:1, v/v), DMSO:ACN (1:9, v/v) or isopropanol was investigated in the SPCL assay buffer by mixing 250 µL of the 100 mM test item solution with ammonium acetate buffer pH 10.2 followed by vortex mixing. For all four solvents, formation ofa phase separation in the form of an oily top layer was observed.

As ACN is the preferred solvent for the DPRA, this solvent was used to dissolvethe test itemin this DPRA study.

Cysteine Reactivity Assay

The reactivity of Hexyl Butyrate towards SPCC was determined by quantification of the remaining concentration of SPCC using HPLC-PDA analysis, following 24.5 hours of incubation at 25±2.5°C. Representative chromatograms of CCcys-209412/Aand209412/A‑cys samples are presented inAppendix 4. An overview of the retention time at 220 nm and peak areas at 220 nm and 258 nm are presented inTable 3(Appendix 3).

Acceptability of the Cysteine Reactivity Assay

The SPCC standard calibration curve is presented inFigure 1(Appendix 2). The correlation coefficient (r²) of the SPCC standard calibration curve was 0.998. Since the r² was >0.99, the SPCC standard calibration curve was accepted.

The results of the Reference Control samples A and C are presented in Table 4 (Appendix 3). The mean peptide concentration of Reference Controls A was 0.503 ± 0.005 mM while the mean peptide concentration of Reference Controls C was 0.508 ± 0.005 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion.

The SPCC peak areas for Reference controls B and C are presented inTable 5(Appendix 3). The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 1.3%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

The SPCC A₂₂₀/A₂₅₈ area ratios of Reference controls A, B and C are presented in Table 6 (Appendix 3). The mean area ratio (A₂₂₀/A₂₅₈) of the Reference Control samples was 19.84. The mean A₂₂₀/A₂₅₈ ratio± 10% range was 17.85-21.82. Each sample showing an A₂₂₀/A₂₅₈ ratio within this range gives an indication that co-elution has not occurred.

The results of the positive control cinnamic aldehyde are presented in Table 7(Appendix 3). The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 74.5% ± 0.9%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

Results Cysteine Reactivity Assay for the Test Item

Preparation of a 100 mMHexyl Butyratestock solution in ACN showed thatthe test itemwas dissolved completely. Upon preparation and after incubation, both the CC as well as the test item samples were visually inspected. Upon preparation as well as after incubation a phase separation in the form of an oily top layer was observed in the co-elution control (CC) and test item samples. In this case one cannot be sure how much test item remained in the solution to react with the peptide.

The results of the cysteine reactivity assay for thetest itemare presented inTable 8(Appendix 3). In the CC sample no peak was observed at the retention time of SPCC (see chromatogram in Appendix 4). This demonstrated that there was no co-elution of the test item with SPCC. For the209412/A-cys samples, the mean SPCC A₂₂₀/A₂₅₈area ratio was 20.10. Since his was within the 17.85-21.82 range, this again indicated that there was no co-elution ofthe test item with SPCC.

The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the test item was 0.6%± 0.6%.

Lysine Reactivity Assay

The reactivity of Hexyl Butyrate towards SPCL was determined by quantification of the remaining concentration of SPCL using HPLC-PDA analysis, following 24.5 hours of incubation at 25±2.5°C. Representative chromatograms of CClys-209412/Aand209412/A‑lys samples are presented in Appendix 4. An overview of the retention time at 220 nm and peak areas at 220 nm and 258 nm are presented in Table 9 (Appendix 3).

Acceptability of the Lysine Reactivity Assay

The SPCL standard calibration curve is presented inFigure 2(Appendix 2). The correlation coefficient (r²) of the SPCL standard calibration curve was 0.996. Since the r²was >0.99, the SPCL standard calibration curve was accepted.

The results of the Reference Control samples A and C are presented inTable 10 (Appendix 3). The mean peptide concentration of Reference Controls A was 0.505 ± 0.009 mM while the mean peptide concentration of Reference Controls C was 0.501 ± 0.010 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolvethe test itemdid not impact the Percent SPCL Depletion.

The SPCL peak areas for Reference controls B and C are presented in Table 11(Appendix 3). The CV of the peptide areas for the nine Reference Controls B and C was 1.5%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

The SPCL A₂₂₀/A₂₅₈ area ratios of Reference controls A, B and C are presented inTable 12 (Appendix 3). The mean area ratio (A₂₂₀/A₂₅₈) of the Reference Control samples was 18.37. The mean A₂₂₀/A₂₅₈ ratio ± 10% range was 16.53-20.21. Each sample showing an A₂₂₀/A₂₅₈ ratio within this range gives an indication that co-elution has not occurred.

The results of the positive control cinnamic aldehyde are presented in Table 13 (Appendix 3). The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 54.8% ± 1.4%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Results Lysine Reactivity Assay for the Test Item

Preparation of a 100 mM Hexyl Butyratestock solution in ACN showed that thetest itemwas dissolved completely. Upon preparation and after incubation, both the CC as well as the test item samples were visually inspected. Upon preparation as well as after incubation a phase separation in the form of an oily top layer was observed in the CC andthetest item samples. In this case one cannot be sure how much test item remained in the solution to react with the peptide.

The results of the lysine reactivity assay forthe test itemare presented inTable 14(Appendix 3). In the CC sample no peak was observed at the retention time of SPCL (see chromatogram in Appendix 4). This demonstrated that there was no co-elution of the test item with SPCL. For the209412/A-lys samples, the mean SPCL A₂₂₀/A₂₅₈ area ratio was 18.37. Since this was within the 16.53-20.21 range, this again indicated that there was no co-elution ofthe test item with SPCL.

The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the Test Item was 0.4%± 0.5%.

DPRA Prediction and Reactivity Classification

Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a phase separation was observed.

An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table below. In the cysteine reactivity assay the test item showed 0.6% SPCC depletion while in the lysine reactivity assay the test item showed 0.4% SPCL depletion. The mean of the SPCC and SPCL depletion was 0.5% and as a result the test item was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

 

SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification forthe Test Item

Test item	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	DPRA prediction and reactivity classification
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
Hexyl Butyrate	0.6%	±0.6%	0.4%	±0.5%	0.5%	Negative: No or minimal reactivity

SD = Standard Deviation.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, this DPRA test is valid. Hexyl Butyrate was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since phase separation was observed after the incubation period for both SPCC and SPCL, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care.

Executive summary:

In this guideline (OECD 442C) study, performed with GLP certification, the test substance Hexyl Butyrate (EC 220-136-6) was found to not be classified as a skin sensitiser under Regulation EC 1272/2008.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 May 2018 to 08 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

This is a Non in-vivo test and the test material is used in cosmetic ingredients. Regulation 1223/2009 Article 18 restricts the use of in-vivo studies on these types of raw materials.

Specific details on test material used for the study:

Appearance: Clear colourless liquid
Purity/Composition: 99.26%, assumed 100% for test
Test item storage: At room temperature protected from light

Details on the study design:

The study was conducted to investigate the potential of the test material to induce genes that are regulated by the antioxidant response element (ARE). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
The ARE-Nrf2 luciferase test method utilises an immortalised adherent cell line derived from HaCaT human keratinocytes. The cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 promoter fused with the ARE from a gene known to be up-regulated by contact sensitisers.
The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by
luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic substances.

Specifications
KeratinoSens™ cell line supplied by Givaudan Schweiz, Duebendorf, Switzerland.
Identification
The test system was appropriately labelled with the study number, assay type, experiment number and test/positive/negative control.
Preparation of Cultures
A fresh vial of cells was used for each experimental occasion and cultured using Dulbecco’s modified Eagle medium (DMEM) containing serum and Geneticin.
Treatment Plate Preparation
The cells were 80-90% confluent (see Section 9 for details of protocol deviations). On the day prior to treatment, cells were harvested and distributed into 96-well plates (10000 cells/well) and incubated at 37±1°C, 5% (v/v) CO2, for 24±1 hours.
For each repetition, three replicates were used for the luciferase activity measurements and one parallel replicate used for the cell viability assay.
Treatment
At the end of the 24-hour incubation period, the medium was removed and replaced with fresh culture medium (containing serum but without Geneticin) to which test article and control formulations were added.
One well per plate was left empty (no cells and no treatment) to assess background values.
Each plate was sealed and incubated at 37±1°C, 5% (v/v) CO2 in air, in a humidified environment for 48±1 hours.
For each test article and positive control, one experiment was needed to derive a prediction (positive or negative), consisting of at two independent repetitions each containing three replicates of each concentration.
The data for repetition 1 was obtained from a repeat experiment as the initial experiment did not meet the acceptance criteria for the positive or negative controls.
The data from the initial experiment has not been reported.
Discordant results were obtained between the two repetitions, therefore a third repetition containing three replicates was performed.
Each independent repetition was performed on a different day with fresh stock solutions of chemicals and independently harvested cells. The cells came from different passages.
Cytotoxicity Assessment
After the 48-hour exposure period, the medium was replaced with fresh medium containing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).The plate was sealed and incubated for 4 hours at 37±1°C, 5% (v/v) CO2.
The MTT medium was removed and SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator at 37±1°C, 5% (v/v) CO2 in air and left overnight.
After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm using a SpectraMax M2e.

Luciferase Activity Measurements
After the 48-hour exposure period, the cells were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plates were then incubated for 20 minutes at 25±2°C, loaded into the luminescence plate
reader and read using the following parameters: 100 µL injection (Luciferase assay substrate), 15 second delay, 7 second luminescence integration time.

Positive control results:

Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.64 and the EC1.5 79 µM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.49 and the EC1.5 51 µM.

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: EC1.5

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: EC1.5

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable

Run / experiment:

other: Experiment 1

Parameter:

other: Imax

Value:

1.1

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Experiment 2

Parameter:

other: Imax

Value:

1.09

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Experiment 1

Parameter:

other: IC30

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable

Run / experiment:

other: Experiment 2

Parameter:

other: IC30

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable

Run / experiment:

other: Experiment 1

Parameter:

other: IC50

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable

Run / experiment:

other: Experiment 2

Parameter:

other: IC50

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable

Hexyl Butyrate was evaluated for the ability to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway. An overview of the viability and luciferase activity induction is summarized in Table 1 and Figure 2-3. The results of the positive control are summarized in Table 2 and Figure 4-5. An overview of EC_1.5, I_max, IC₃₀ and IC₅₀ values is given in Table 3. The individual raw data are presented in Appendix 3 and Appendix 4. The historical control data are presented in Appendix 5.

Two independent experiments were performed. The cells were in these experiments incubated withHexyl Butyratein a concentration range of 0.98 - 2000 µM (2-fold dilution steps) for 48 hours ±1h. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay.

 

Experiment 1

·          No precipitation was observed at the start and end of the incubation period in the 96-well plates.

·          Hexyl Butyrate showed no toxicity. The viability of the cells was higher than 70% at all test concentrations except in the mid dose level of 500 µM where a viability was observed of 37%. This sudden drop in viability was not dose related and not reproducible in the second experiment and therefore no IC₃₀ and no IC₅₀ was calculated. 

·          No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with Hexyl Butyrate. The I_max was 1.10 and therefore no EC_1.5 could be calculated. 

·          The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The I_max was 2.64 and the EC_1.5 79 µM.

Experiment 2

·          No precipitation was observed at the start and end of the incubation period in the 96-well plates.

·          Hexyl Butyrate showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC₃₀and IC₅₀values could be calculated. 

·          No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with Hexyl Butyrate. The I_maxwas 1.09 and therefore no EC_1.5 could be calculated. 

·          The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The I_max was 2.49 and the EC_1.5 51 µM.

Both tests passed the acceptance criteria:

·          The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration. 

·          The EC_1.5 of the positive control was between 5 and 125 µM (79 µM and 51 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.64-fold and 2.49-fold in experiment 1 and 2, respectively).

·          Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (8.8% and 13% in experiment 1 and 2, respectively).

 

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, Hexyl Butyrate is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the ability of Hexyl Butyrate to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens(TM) assay.

The study procedures described in this report were based on the most recent OECD guideline.

Batch 202509 of Hexyl Butyrate was a clear colourless liquid. Hexyl Butyrate was dissolved in dimethyl sulfoxide at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 µM (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. No precipitate was observed at any dose level tested. Two independent experiments were performed.

Both experiments passed the acceptance criteria:

• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.
• The EC_1.5 of the positive control was between 5 and 125 µM (79 µM and 51 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.64-fold and 2.49-fold in experiment 1 and 2, respectively).
• Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (8.8% and 13% in experiment 1 and 2, respectively).

 

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Hexyl Butyrate showed no toxicity (no IC₃₀ and IC₅₀ value) in both experiments except in the first experiment in the mid dose level of 500 µM where a viability was observed of 37%. This sudden drop in viability was not dose related and not reproducible in the second experiment and therefore no IC₃₀ and no IC₅₀ was calculated. No biologically relevant induction of the luciferase activity (no EC_1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.10-fold and 1.09-fold in experiment 1 and 2 respectively. Hexyl Butyrate is classified as negative in the KeratinoSens(TM) assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 µM.

In conclusion, Hexyl Butyrate is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)